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B Rapoport

Publications and source records attributed to B Rapoport.

At least 19 recordsLinked to original sources
PubMed1996

Structural studies of human autoantibodies. Crystal structure of a thyroid peroxidase autoantibody Fab.

The three-dimensional structure of the Fab of TR1.9, a high-affinity IgG1, kappa human autoantibody to thyroid peroxidase, was determined crystallographically to a resolution of 2.0 A. The combining site was found to be relatively flat, like other antibodies to large proteins. Sequence differences from the most closely related germline genes mainly occur at positions occupied by residues with outward-pointing side chains. An increased deformability of the second and third complementarity-determining regions of the heavy chain may result from the replacement of two germline asparagines and the presence of several glycines, and may allow "induced fit" in the binding to antigen. Four exposed charged residues, resulting from the use of a particular D (diversity) and J (joining) segments in the assembly of the heavy chain, may contribute to the high affinity of antigen binding. The crystal structure of TR1.9 Fab is the first for a human IgG high-affinity autoantibody.

Amino Acid Sequence↗
PubMed1996

Thyroid peroxidase autoantibody epitopic 'fingerprints' in juvenile Hashimoto's thyroiditis: evidence for conservation over time and in families.

In Hashimoto's thyroiditis, the humoral component is manifest by autoantibodies to thyroid peroxidase (TPO). Epitopic 'fingerprinting' of polyclonal serum TPO autoantibodies has been facilitated by the molecular cloning and expression as Fab of a repertoire of human TPO autoantibody genes. To investigate whether TPO autoantibody fingerprints are (i) stable over long periods of time (approximately 15 years), and (ii) inherited, we studied a cohort of nine patients with juvenile Hashimoto's thyroiditis and 21 first degree relatives of four of these patients. Fingerprints were determined by competition between four selected FAB and serum autoantibodies for binding to 125I-TPO. Regardless of titre, the TPO epitopic profile was stable in 10/12 individuals whose TPO autoantibody levels were sufficient for analysis on two or three occasions over 12-15 years. Although the TPO epitopic fingerprint profiles in two families raised the possibility of inheritance, overall the data from all four families did not reveal an obvious pattern of genetic control. In no family was the TPO epitopic fingerprint associated with HLA A, B or DR. In conclusion, TPO autoantibody epitopic fingerprints are frequently conserved over many years. Studies on additional families are necessary to establish whether or not the epitopic profiles of TPO autoantibodies are inherited.

Adolescent↗
PubMed1996

The quest for the autoantibody immunodominant region on thyroid peroxidase: guided mutagenesis based on a hypothetical three-dimensional model.

Thyroid peroxidase (TPO), a cell surface glycoprotein, is the major autoantigen in autoimmune thyroiditis in humans. The molecular cloning and expression of Ig genes from thyroid-infiltrating B cells has generated a large repertoire of human TPO Fab that have been used to map an immunodominant region on TPO. However, the topological site of this region, consisting of a cluster of highly conformational epitopes, remains unknown. Using the recently elucidated three-dimensional structure of myeloperoxidase as a model, we stably expressed on the surface of eukaryotic cells eight "guided" mutants of the TPO molecule. The sites of these mutations were strategically located to alter the surface contour of the molecule with minimal disruption to its core structure. Remarkably, in the present study (in contrast to previous unguided TPO mutagenesis studies), all eight TPO mutants retained recognition by the TPO Fab. These results support the validity of the model used for mutagenesis. Although not identifying the immunodominant region on TPO in thyroid autoimmunity, our data provide evidence against the involvement of certain topological segments and may help to narrow the search for this region. The most open region remaining as a candidate location is the antero-inferior portion of the molecule.

Amino Acid Sequence↗
PubMed1996

Critical relationship between autoantibody recognition and thyrotropin receptor maturation as reflected in the acquisition of complex carbohydrate.

Generation of large amounts of recombinant TSH receptor (TSHR) protein capable of recognition by TSHR autoantibodies is a goal of clinical importance. We expressed in Chinese hamster ovary cells the human TSHR ectodomain (ECD) with a carboxyl-terminus six-histidine tag. After transgene amplification, expressing clones were selected by nickel chelate chromatography in combination with [35S] methionine precursor labeling. An approximately 74-kDa protein was detected in the culture medium, and larger quantities of an approximately 68-kDa protein were found in the cell soluble fraction. Immunoblot analysis with a rabbit antiserum revealed that most of the TSHR-ECD was not secreted, but was retained within the soluble fraction of the cell. Nickel chelate chromatography was not effective in purifying significant quantities of this material. In contrast, with Concanavalin A, but not with wheat germ agglutinin, an approximately 50-fold purification of TSHR-ECD was achieved from the cell soluble fraction. Surprisingly, this affinity-enriched TSHR, containing high mannose carbohydrate, was not recognized by human TSHR autoantibodies in sera from six individuals. By ion exchange chromatography, the autoantibody-neutralizing TSHR in the cell supernatant fraction was found to be nonidentical with TSHR-ECD protein recognized by antisera from immunized animals. The present data indicate the critical relationship between autoantibody recognition and TSHR maturation as reflected in the acquisition of complex carbohydrate. Nonsecretion of the TSHR-ECD appears to be related to the specific protein rather than to the glycosylation apparatus of the host cell. Antibodies from immunized animals may be ineffective in monitoring purification of autoantigen-competent TSHR. Finally, the data explain why soluble recombinant TSHR generated in many expression systems is not recognized satisfactorily by human autoantibodies.

Animals↗
PubMed1995

Expression of the extracellular domain of the thyrotropin receptor in the baculovirus system using a promoter active earlier than the polyhedrin promoter. Implications for the expression of functional highly glycosylated proteins.

Conventional baculovirus vectors that utilize the very late polyhedrin promoter have not proved successful for expressing a thyrotropin (TSH) receptor capable of ligand and Graves' disease autoantibody binding comparable to the receptor produced in mammalian cells. Because of the clinical importance of high level expression of this protein, we reassessed the baculovirus system using a new transfer vector (pAcMP3) containing the late basic protein promoter, which functions earlier than the classical polyhedrin promoter. Maximal synthesis of the [35S]methionine-labeled TSH receptor extracellular domain, affinity-purified using a 6-histidine tag, occurred earlier (1 day after insect cell infection) than with a vector (pVL1393) containing the polyhedrin promoter. The pAcMP3-derived TSH receptor extracellular domain was larger (approximately 68 kDa) than the pVL1393-derived protein (approximately 63 kDa). Only the 68-kDa product was secreted, albeit in trace amounts detectable only by precursor labeling. Enzymatic deglycosylation reduced both 68- and 63-kDa cellular proteins to approximately 54 kDa, indicating that the pAcMP3 vector generated a protein with greater carbohydrate content. However, despite its greater degree of glycosylation, most of the 68-kDa protein remained within the cell, almost entirely in the particulate fraction. Remarkably, the trace amounts of 68-kDa receptor protein affinity-purified from the soluble cytosolic fraction of infected insect cells completely neutralized TSH receptor autoantibodies in patients' sera and partly inhibited TSH binding. In conclusion, a baculovirus vector with a promoter active earlier than the conventional polyhedrin promoter generates a more glycosylated and functional TSH receptor extracellular domain protein, albeit at low levels. These data carry important implications for the expression by baculovirus vectors of functional, highly glycosylated proteins.

Animals↗
PubMed1995

Thyrotropin binding specificity for the thyrotropin receptor.

Recently, highly purified bovine thyrotropin (bTSH) of pituitary origin, as well as recombinant human (h) TSH free of lutropin (LH) contamination, has been reported to activate the LH/choriogonadotropin receptor (LH/CGR). These data challenge the concept of TSH specificity for its own receptor. We, therefore, re-evaluated these data using, as targets, the recombinant hTSH and rat LH/CGRs stably expressed in Chinese hamster ovary (CHO) cells. Partially purified bTSH (2 IU/mg protein) and, to a lesser degree, highly purified bTSH (30 IU/mg protein) increased intracellular cAMP levels in CHO-LH/CGR cells (an EC50 of 0.2 and > 20 mIU/ml, respectively). In contrast, recombinant hTSH (up to 1 IU/ml) did not. All three TSH preparations increased cAMP levels to the same extent in CHO-TSHR cells (an EC50 of 0.3 mIU/ml). Furthermore, we observed only nonspecific, low affinity TSH binding for CHO-LH/CGR cells and also for CHO cells transfected with the expression vector alone (a Kd of 100 nM), although both high and low affinity TSH binding was demonstrated in CHOT-SHR cells (a Kd of 0.3 and 100 nM, respectively). These data indicate that even highly purified bTSH of pituitary origin contains significant amounts of LH, and that TSH itself does not appear to activate the LH/CGR.

Animals↗
PubMed1995

Molecular cloning and characterization of human thyroid peroxidase autoantibodies of lambda light chain type.

IgG class thyroid peroxidase (TPO) autoantibodies with kappa light (L) chains predominate in serum and the genes for a large repertoire of such autoantibodies have been characterized. The present study was performed to clone and characterize TPO autoantibodies with lambda L chains which comprise approximately 20% of serum TPO autoantibodies. From a combinatorial IgG H/lambda L chain cDNA library in the phage display vector pComb3, 24 TPO-binding clones with lambda L chains were isolated, comprising three different heavy (H) and light (L) chain combinations. These combinations utilized two genes from the Vlambda II and IIIb families (closest germline genes DPL11 and hsigg11150) and three genes from the VH1, VH3 and VH4 families (VH26, 4.34 and hv1L1). The deduced amino acid sequences of these H chains were quite different from those of kappa F(ab) isolated using the same H chain library. We expressed the proteins for these three lambda F(ab), as well as for a lambda F(ab) (Humlv318 L chain/DP10-like H chain) previously isolated from another patient. The affinities for TPO of the lambda F(ab) (Kd 8 x 10(-10) M to 10(-7) M) were lower than those of the kappa F(ab) (Kd approximately 10(-10) M). For two lambda F(ab), both H and L chain genes were close to germline configuration, but there was no straightforward relationship between the extent of somatic mutation from germline configuration and affinity for TPO. All four lambda F(ab) bound less well to denatured TPO as to native TPO. The three F(ab) for which sufficient protein could be expressed for competition studies all recognized domain B within the immunodominant region on TPO previously identified using F(ab) with kappa L chains. Aside from these TPO-specific F(ab), only a few other human IgG class, organ-specific autoantibodies with lambda L chains have been characterized at the molecular level. Our study significantly augments the small database on this category of autoantibodies in general.

Amino Acid Sequence↗
PubMed1995

Combinatorial libraries: new insights into human organ-specific autoantibodies.

The recent application of immunoglobulin (Ig) gene combinatorial library technology has led to a logarithmic increase in information concerning human, disease-associated, organ-specific autoantibodies of the IgG class. As reviewed here by Basil Rapoport, Stefano Portolano and Sandra McLachlan, the molecular cloning, analysis and expression of the genes for increasing numbers of these human, monoclonal autoantibodies is providing new insight into the genetic background and epitopic repertoires of such molecules.

Antibodies, Monoclonal↗
PubMed1995

Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response.

TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with VH gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production.

Animals↗
PubMed1995

Thyroid peroxidase autoantibody fingerprints. II. A longitudinal study in postpartum thyroiditis.

It is not known whether epitopes recognized by autoantibodies in an individual remain constant or change over time, especially during perturbations of the humoral immune response. To address this question, we studied the epitopic profile ("fingerprint") of autoantibodies to thyroid peroxidase (TPO) in the sera of 19 women during the postpartum period. Fingerprints were determined in competition studies using 4 recombinant F(ab). At delivery and at 3 time intervals over the subsequent 9-12 months, the pool of F(ab) inhibited autoantibody binding to TPO by 80-100%, consistent with the definition by these F(ab) of a TPO immunodominant region (A1, A2, B1, and B2 domains). Despite a wide spectrum among individuals, the TPO epitopic fingerprints for all 19 women were relatively unchanged throughout the postpartum period. Fingerprint constancy occurred regardless of fluctuations in serum TPO autoantibody levels. When assessed numerically as a ratio of inhibition by the A domain F(ab) to inhibition by the B domain F(ab), the A/B domain ratios in individual women ranged from 0.2 (predominantly B domain) to more than 3.0 (predominantly A domain). However, for each individual woman, the A/B epitopic ratio was conserved throughout the study interval. Our TPO autoantibody epitopic fingerprint data have potential implications for understanding the humoral autoimmune response in man. First, the present study indicates a remarkable lack of spreading of B cell epitopes during a state of perturbation of the immune system over a period of 1 yr. Second, and perhaps more important, despite marked variations in TPO epitopic profiles among different individuals, their constancy over time suggests that TPO autoantibody fingerprints may be inherited.

Autoantibodies↗
PubMed1995

Thyroid peroxidase autoantibody fingerprints in hypothyroid and euthyroid individuals. I. Cross-sectional study in elderly women.

Human monoclonal immunoglobulin G-class autoantibodies to thyroid peroxidase (TPO), expressed as recombinant F(ab), are powerful tools for analyzing the individual components of polyclonal serum TPO autoantibodies. Four TPO-specific F(ab) interact with epitopes in two closely related domains (A and B) in the immunodominant region on TPO. In the present study, these TPO F(ab) were used to compete for serum autoantibody binding to [125I]TPO to determine the "epitopic fingerprints" in two groups of carefully controlled individuals. All individuals (14 hypothyroid and 32 euthyroid) were elderly women (60-71 yr old) with similar genetic and environmental backgrounds as well as comparable levels of serum TPO autoantibodies. Using the pool of four F(ab), serum TPO autoantibody binding was inhibited to the same extent (approximately 90%) in hypothyroid and euthyroid individuals, demonstrating that the majority of TPO autoantibodies in both groups recognize the TPO immunodominant domain. When tested individually, the F(ab) produced a spectrum of inhibition patterns, ranging from sera preferentially inhibited by domain A F(ab) to sera preferential inhibited by domain B F(ab). The ratio of inhibition by domain A F(ab) to inhibition by domain B F(ab) was similar in hypothyroid (0.11-1.39) and euthyroid (0.21-1.79) women. In conclusion, no difference in TPO autoantibody epitopes was observed in this cross-sectional study of hypothyroid and euthyroid individuals. Longitudinal studies are required to address the question of whether TPO autoantibody epitopic fingerprints are stable over time.

Adult↗
PubMed1995

Genetic alterations in thyroid hyperfunctioning adenomas.

Thirty-seven thyroid autonomously hyperfunctioning adenomas were screened for mutations in the TSH receptor (TSHR), G alpha s (gsp), and ras genes. Polymerase chain reaction-amplified fragments of the TSHR C-terminal part (exon 10), the G alpha s (exons 8 and 9), and the three ras genes were obtained from the genomic DNA extracted from 37 tumors and their adjacent normal tissues and were studied by direct nucleotide sequencing and hybridization with synthetic probes. A point mutation in the third intracellular loop (codon 623) of the TSHR was found in 3 of 37 adenomas studied. This mutation codes for a change (Ala to Ser) in the TSHR structure and is somatic and heterozygotic. Constitutive activation of the TSHR was demonstrated by an increase in basal cAMP levels after transfection of Chinese hamster ovary cells with a mutated Ser623-TSHR complementary DNA. Nine gsp[00ae]MDRV[00af]- and one ras-activating mutations were also detected. No simultaneous alteration of the studied genes was present. Thus, in hyperfunctioning thyroid adenomas, our data suggest that a mutational activation of the TSHR and gsp genes may play a tumorigenic role through constitutive activation of the cAMP pathway.

Adenoma↗
PubMed1995

Human monoclonal autoantibodies against the immunodominant region on thyroid peroxidase: lack of cross-reactivity with related peroxidases or thyroglobulin and inability to inhibit thyroid peroxidase enzymatic activity.

Thyroid peroxidase (TPO) autoantibodies are heterogeneous and have been classified in terms of whether they cross-react with myeloperoxidase (MPO), lactoperoxidase (LPO), or thyroglobulin (Tg) as well as by whether they inhibit TPO enzymatic activity. Four human monoclonal TPO autoantibodies, generated using combinatorial immunoglobulin gene libraries and expressed as F(ab), have been used to investigate these properties of TPO autoantibodies. The binding of F(ab) WR1.7, TR1.8, TR1.9, and SP1.4 to 125I-labeled recombinant TPO was inhibited 50% by approximately 10(-10) mol/L unlabeled TPO, reflecting the high affinities of these F(ab) for TPO. In contrast, F(ab) binding to TPO was unaffected by human MPO (both native and reduced), bovine LPO, or human Tg at concentrations up to 10(-8) mol/L. Further, TPO enzymatic activity, measured by guiacol oxidation, was unaffected by preincubation with the four F(ab) individually or as a pool (each at 10(-8) mol/L). In conclusion, four human TPO monoclonal autoantibodies do not cross-react with related peroxidases or Tg, nor do they inhibit TPO enzymatic activity. These monoclonal immunoglobulin G class autoantibodies define the immunodominant region on TPO and represent about 85% of TPO autoantibodies in an individual patient's serum. Consequently, our data suggest that TPO autoantibodies that cross-react with MPO, LPO, or Tg, or inhibit TPO enzymatic activity are likely to bind outside the immunodominant region.

Antibodies, Monoclonal↗
PubMed1994

Cleavage of the thyrotropin receptor does not occur at a classical subtilisin-related proprotein convertase endoproteolytic site.

The human thyrotropin receptor (TSHR) undergoes proteolytic cleavage closely upstream to amino acid 317. Between residues 261 and 313 are three clusters of positively charged amino acids, arginines (Arg) and lysines (Lys), which are potential subtilisin-related proprotein convertase sites. We used oligonucleotide-directed mutagenesis to perform conservative amino acid substitutions within these regions (Arg or Lys to glutamine, Gln). Chinese hamster ovary cells stably transfected with mutant receptor cDNA TSHR-CS1 (Gln261) and TSHR-CS3 (Gln312, Gln313) bound radiolabeled TSH with an affinity similar to the wild-type TSHR. Mutant cDNA TSHR-CS2 (Gln290, Gln291) and TSHR-CS4 (Gln261, Gln290, Gln291, Gln312, Gln313) did not express a protein on the cell surface capable of specific TSH binding. After covalent cross-linkage of radiolabeled TSH to TSHR-CS1 and TSHR-CS3, the mutant receptors dissociated into two subunits under reducing conditions. The most prominent cluster of basic amino acids in the TSHR extracellular region (residues 287-293) was studied in a second series of mutations designed to eliminate the classical proprotein convertase sites in this region and yet be compatible with TSHR function. All three mutant receptors, TSHR-CS5 (Gln290), TSHR-CS6 (Gln291), and TSHR-CS7 (Gln291, Gln293) bound TSH with an affinity similar to that of wild type, and none of these amino acid substitutions prevented proteolytic cleavage of the extracellular domains of the TSHR. Thus, cleavage of the TSHR extracellular domain does not involve a classical subtilisin-related proprotein convertase cleavage site, raising the possibility that TSHR cleavage occurs after processing and trafficking of the protein to the plasma membrane.

Amino Acid Sequence↗
PubMed1994

Immunoglobulin A in Graves' orbital tissue: deoxyribonucleic acid amplification by polymerase chain reaction.

A role for IgA autoantibodies in Graves' ophthalmopathy is suggested by the presence of immunoglobulins of this class in Graves' orbital tissue, as detected by immunohistochemistry. We, therefore, investigated the possibility of using the polymerase chain reaction (PCR) to amplify IgA immunoglobulin genes from plasma cells infiltrating Graves' eye tissue. Template cDNA was reverse-transcribed from orbital muscle (M) mRNA of one patient (#7) and from orbital connective tissue/fat (F) mRNA of two patients (#1 and #7), both undergoing surgery for exophthalmos because of severe infiltrative ophthalmopathy. Preliminary studies to establish the PCR procedure were performed for kappa light chain DNA amplification. With the very small amount of orbital tissue template available, the sensitive "hot start" modification of the PCR was necessary to amplify significant amounts of kappa light chain DNA. Using this procedure, IgA heavy chain DNA was amplified from both connective tissue/fat (F7) and muscle (M7) cDNA of patient #7. The DNA yield was less for IgA than for IgG using the same template. There was no significant IgA (or IgG) DNA product using the connective tissue/fat cDNA of patient #1. While not implying that IgA-infiltrating plasma cells are specific for Graves' orbital tissue, our studies nevertheless demonstrate the feasibility of amplifying the genes coding for IgA antibodies from Graves' orbital tissue plasma cells. Expression of these immunoglobulin genes in future studies will make it possible to determine the antigen specificity of the antibodies expressed by Graves' orbital tissue plasma cells.

Aged↗
PubMed1994

Profile of lambda light chain variable region genes in Graves' orbital tissue.

Graves' ophthalmopathy, a human autoimmune disease of unknown etiology, is strongly associated with autoimmune hyperthyroidism. A major controversy is whether retro-ocular muscle or orbital fat/connective tissue is the target of the immune response. Previously, we observed preferential PCR amplification of lambda (relative to kappa) light chain DNA from cDNA of Graves' orbital tissue-infiltrating B cells/plasma cells. There is little information on V lambda gene usage in man and none in diseased tissue. To characterize the orbital lambda light chains, we constructed cDNA libraries using PCR-amplified DNA from three tissues and sequenced the variable region genes from randomly selected clones. Analysis of 27 clones from orbital fat/connective tissue libraries from two patients with acute inflammatory eye disease, and 15 clones from orbital muscle of one of these patients, revealed a diverse spectrum of lambda V region genes. The nucleotide sequences of these 42 clones were most homologous to 12 different germline genes: four family I (subfamilies I-a, -b and -c), three family II, two family III and one family VII germline genes. Each orbital tissue had a distinct profile of V lambda sequences. However, all clones used J lambda 2/3 and all three orbital tissues contained clones related to family II genes. Although some clones had V region sequences in near germline conformation, the majority differed from the closest germline gene in both framework and complementarity determining regions. Whether or not these differences result from multiple germline gene usage or somatic mutation of a smaller number of germline genes cannot be determined until information on the V lambda repertoire and its polymorphisms is complete. However, the V lambda gene diversity we observed in both orbital muscle and orbital fat/connective tissue suggests a role for lambda autoantibodies in the pathogenesis of Graves' ophthalmopathy.

Adipose Tissue↗